1. Field of the Invention
This invention relates to transport of compounds into cells, in particular, transport of heterologous compounds such as peptides and proteins, into eukaryotic cells.
2. Description of the Related Art
The plasma membranes of cells present a barrier to passage of many useful therapeutic agents. In general, a molecule must be freely soluble in both the aqueous compartments of the body and the lipid layers through which it must pass, in order to enter cells. Many therapeutic macromolecules such as peptides and oligonucleotides are also particularly intractable to transmembrane transport. Current DNA transfection techniques include microinjection, calcium phosphate co-precipitation, cationic liposomes, viral vectors and electroporation. These methods are capable of transporting DNA into cells, but the techniques are cumbersome and cytotoxic. Furthermore, once transfection has been completed, the researcher must wait 12-80 h after transfection to detect expression of the gene of interest.
Recently, novel methods have been developed for the delivery of exogenous peptides and proteins directly into living cells with the help of protein transduction domains such as HIV-1 Tat (48-60) (A. Frankel and C. Pabo, Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 55 (1988), pp. 1189-1193; M. Green and P. Loewenstein, Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans-activator protein. Cell 55 (1988), pp. 1179-1188). However, this method has drawbacks. Protein transduction via Tat-fusion proteins resulted in inactivation and denaturation of the cargo protein. To deliver an active protein, correct renaturation is required upon internalization. In addition, Tat must be covalently linked by a chemical reaction to the compounds or macromolecules to be delivered.
Another transduction domain protein has been reported from the Drosophila Antennapedia homeotic transcription factor (43-58) (A. Joliot, A. Triller, M. Volovitch, C. Pemelle and A. Prochiantz, Alpha-2-8-polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol 3 (1991), pp. 1121-1134.; I. Le Roux, A. H. Joliot, E. Bloch-Gallego, A. Prochiantz and M. Volovitch, Neurotrophic activity of the antennapedia homeodomain depends on its specific DNA-binding properties. Proc Natl Acad Sci USA 90 (1993), pp. 9120-9124). A commercially available protein transduction domain peptide, PENETRATIN® (U.S. Pat. No. 5,888,762) based upon this peptide consists of 16 amino acids corresponding to the third helix of the homeodomain of antennapedia protein. Activated PENETRATIN® has an N-terminal pyridyl disulfide that covalently couples with cargo peptides. However, chemical coupling is potentially cumbersome and cargo peptides must have a free thiol group for chemical coupling with this reagent.
Another protein transduction domain peptide utilizes the translocation properties of the 38 kDa herpes simplex virus-1 DNA binding protein, VP22 (G. Elliott and P. O'Hare, Intercellular trafficking and protein delivery by a herpes virus structural protein. Cell 88 (1997), pp. 223-233). However, VP22 must be fused to the peptide/protein to be delivered and requires the construction of a suitable expression vector to make the fusion protein.
Rothbard, et al. (U.S. Pat. No. 6,306,993) disclose a method for enhancing transport of a compound across a biological membrane, by conjugation between the transported compound and a transport polymer by covalent attachment. The transport polymer includes subunits, at least 50% of which contain a guanidino or amidino sidechain moiety. U.S. Pat. No. 6,306,993 also requires a covalent attachment of the cargo molecule to the transporter.
For the carrier peptides described above, hybridization of the carrier peptides either genetically or chemically with the molecule to be transported was required for the efficient intracellular delivery of the various cargo peptides and proteins.
WO02/10201 (Divida, et al.) disclose a transfection agent that includes a peptide (15-30 residues) with hydrophobic and hydrophilic domain which provides a non-covalent association with and transport of a heterologous compound into a cell. This agent is commercially available as, CHARIOT™, which delivers biologically active proteins, peptides and antibodies directly into cultured mammalian cells at an efficiency of 60-95%. The CHARIOT™ peptide forms a non-covalent bond with cargo peptides. This may stabilize the protein, protect the cargo protein from degradation and preserve the natural characteristics of the cargo protein during the transfection process. Although covalent attachment is not required, it is necessary to prepare a mixture of CHARIOT™ and the cargo peptides before delivery.
Generally, in order to successfully deliver peptides into desired cells, the protein transduction domains, Tat, VP22 and antennapedia protein, require covalent linkage to the cargo compounds or macromolecules by either chemical reaction or a biological process such as preparation of a recombinant fusion protein. The preparation of the covalently linked cargo peptide is potentially complex, labor-intensive, and time consuming. The preparation of the cargo protein for transport by such methods also inactivates the biological function(s) of the cargo peptide. While the commercially available protein transduction domain peptide, CHARIOT™, does not require a covalent attachment, it is necessary to make a mixture of CHARIOT™ and the cargo peptides before applying to the cells. This mixing time is also potentially time consuming.